Optimization Telomerase Activity Assay for Pinus tabulaeformis Carr.

doi:10.11924/j.issn.1000-6850.2014-0025

Abstract

Abstract: Telomerase is essential to maintain the integrity of eukaryotic chromosomes because of its function for restoring and extending telomere length. Though there are well established methods for measuring telomerase activity in animal cells, telomerase activity assay for plant cells is far away from effective, method to determine the telomerase activity is of great significance. In this study, we adopted and optimized TRAP method to determine the telomerase activity of the P. tabulaeformis. The optimization were made to the steps of the extension condition, the primer, the annealing temperature and the electrophoresis detection. The results showed that the best extension condition was one hour for 30℃, the optimum guide primer was the TS21 and the reverse primer was the ACX, the annealing temperature was 61.8℃, and the Gene Finder nucleic acid dye was selected to stain the polyacrylamide gel, respectively. Based on our data, optimized TRAP assay could effectively detect telomerase activity of the P. tabulaeformis, made it a valuable tool in testing telomerase activity in plant, an effective method for measuring the telomerase activity in P. tabulaeformis was established.

References

[1] Aronen T, Ryynanen L. Variation in telomeric repeats of Scots pine (Pinus sylvestris L.) [J]. Tree Genetics & Genomes,2012,8(2):267- 275.
[2] Aronen T, Pehkonen T, Ryyn?nen L. Enhancement of somatic embryogenesis from immature zygotic embryos of Pinus sylvestris [J]. Scand J Forest Res,2009,24(5):372-383.
[3] Flanary B E, Kletetschka G. Analysis of telomere length and telomerase activity in tree species of various life- spans, and with age in the bristlecone pine Pinus longaeva[J]. Biogerontology,2005,6 (2):101-111.
[4] Osterhage J L, Friedman K L. Chromosome end maintenance by telomerase[J]. Biol Chem,2009,284(24):16061-16065.
[5] Zhang L L, Katsunori T, Kazuo S, et al. The telomerase inhibitor telomestatin induces telomere shortening and cell death in Arabidopsis .BBA,2005,1763(2006):39-44.
[6] Kim N W, Wu F. Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP). Nucleic Acids Res,1997,25(13):2595-2597.
[7] Kim N W, Piatyszek M A, Prowse K R, et al. Specific association of human telomerase activity with immortal cells and cancer. Science, 1994,266(5193):2011-2015.
[8] 陈波,梁江丽,田晓平,等.水稻愈伤组织端粒酶催化特征检测[J].浙江理工大学学报,2009,26(4):579-601.
[9] Fajkus J, Sykorovae, Leitch A R. Telomeres in evolution and evolution of telomeres[J]. Chromosome Res,2005,13(5):469-479.
[10] Song H, Liu D, Li F L, et al. Season- and age-associated telomerase activity in Ginkgo biloba L[J]. Mol Biol Rep,2011,38(3):1799-1805.
[11] 边杉,王颖,于涛,等.聚丙烯酰胺凝胶银染技术在半定量 RT-PCR中的应用[J].中国药科大学学报,2004,35(2):178-182.
[12] 陈波,梁江丽,田晓平,等.水稻愈伤组织端粒酶催化特征检测[J].浙江理工大学学报,2009,26(4):597-601.
[13] 王瑾瑜,张徐俞,王雅群,等.用改进的 TRAP法测定树木端粒酶活性[J].应用与环境生物学报,2012,18(4):682-686.
[14] 吴东林,张玉静,李鹏,等.端粒酶活性检测方法的改进及 dNTP浓度对端粒酶活性的影响[J].中国兽医学报,2002,22(6):585-586.
[15] Oguchi K, Tamura K, Takahashi H. Characterization of Oryza sativa telomerase reverse transcriptase and possible role of its phosphorylation in the control of telomerase activity[J]. Gene,2004, 342(1):57-66.
[16] Fitzgerald M S. Different modes of de novo telomere formationby plant telomerases[J]. Plant Journal,2001,26(1):77-87.
[17] Fitzgerld M S, Mcknight T D, Dorothy E, et al. Characterization and developmental patterns of telomerase expression in plants[J]. PNAS,1996,93(25):14422-14427.
[18] 马登旭,杨力媛,马国兴,等.水稻端粒酶 RNA候选序列的克隆及特征解析[J].浙江理工大学学报,2013,30(4):579-584.
[19] 刘小川,梁永恒,陈波.水稻端粒酶活性定量分析研究[J].中国生物工程杂志,2007,27(5):45-49.
[20] 张玉山,白旭峰.一种简单快速高分辨率的 PAGE 胶显带方法[J].遗传,2008,30(2):251-254.